1. Field of the Invention
The present invention relates to various methods for production of phosphorothioate containing nucleic acids which are generally more nuclease resistant than unsubstituted DNA, RNA, or oligonucleotides. Micro-organisms or cells are used to catalyze the in vivo synthesis of phosphorothioate nucleic acids by incubation in modified media containing thio-phosphate as a source of phosphorus. The method enables the bulk preparation of nuclease resistant double-stranded (ds) DNA, single-stranded (ss) DNA, RNA, or oligo mixtures substituted with phosphorothioate linkages.
Bacteria infected with recombinant phage DNA and grown in thio-phosphate containing media produce single-stranded phage DNA with phosphorothioate linkages. Recombinant phosphorothioate phage DNA can be further processed in vitro to generate oligo mixtures spanning the entire region of a cloned recombinant DNA insert. Such oligo mixtures are not only nuclease resistant but also have enhanced antisense activity by virtue of multiple target sites embodied in an entire recombinant cDNA or exon phage DNA insert. Phosphorothioate containing DNA plasmids or recombinant DNA vectors can also be prepared. The use of thiophosphate in generating phosphorothioate nucleic acids in vivo is not limited by cell type as both prokaryotic and eukaryotic cells incorporate the modified phosphate.
2. Description of Related Disclosures
Phosphorothioate containing analogues of oligonucleotides are widely used for gene ablation, otherwise known as antisense technology, to diminish gene expression in tissue culture cells or in the treatment of entire organ systems as novel pharmaceutical reagents (J. Murray Ed. (1992) “Antisense RNA & DNA,” Wiley-Liss, New York; van der Krol, et al (1988) BioTechniques 6:958–975; Clercq, et al (1970) Virology 42:421–428). DNA and RNA oligonucleotides can be chemically synthesized in several hundred milligram to gram quantities (Froehler et al (1986) Nucleic Acids Res. 14:5399–5407; Sinha et al (1983) Tetrahedron Lett. 24:58435846; Letsinger et al (1965) J. American Chem. Soc. 87: 3526; Sinha and Fry (1984) In Sanghui and Cook (Eds) “Carbohydrate Modifications in Antisense Research,” American Chemical Society, Washington, D.C.), although they are often used on a smaller scale for research purposes. Phosphorothioate oligos are typically prepared via solid phase synthesis and oxidative sulfurization with the Beaucage or Zon reagent. More recent advances in include the preparation of chirally enriched phosphorothioate oligos (Just et al (2000) U.S. Pat. No. 6,031,092; Stec et al (1999) U.S. Pat. No. 5,883,237) as well as large scale synthesis procedures involving solution phase techniques (Yau (1997) U.S. Pat. No. 5,644,048; Ravikumar et al (1999) U.S. Pat. No. 6,001,982).
Long phosphorothioate polymers (>100 bases) can be generated in vitro using RNA or DNA polymerases and the appropriate phosphorothioate analogues of the nucleoside triphosphates as substrates (Griffiths and Eperon (1987) Nucleic Acids Res. 15:4145–4162; Suh and Eperon (1987) Nucleic Acids Res. (1992) 20:6303–6309; Eckstein (1985) Annu. Rev. Biochem. 54:367–402). Enzymatic synthesis being more costly, is typically used when smaller quantities suffice or longer polymers are required. Enzymatic synthesis results in the stereospecific incorporation of Sp nucleotides with inversion of configuration to the Rp form in the internucleotidic linkage. In this regard, oligodeoxynucleotides of the Rp configuration form a more stable complex with mRNA and hence may be a better ablater of gene expression (Koziolkiewicz et al (1995) Nucleic Acids Res. 23: 5000–5005). Several novel in vitro approaches have been devised for the enzymatic preparation of oligonucleotides involving the formation of concatamers and various means of separating these (Lackey et al (1998) U.S. Pat. No. 5,739,311; Kacian et al (1999) U.S. Pat. No. 5,916,777; Dattagupta et al (1999) U.S. Pat. No. 5,932,450; Kool et al (2000) U.S. Pat. No. 6,096,880).
The utility of antisense oligos for the ablation of gene expression has gained considerable experimental evidence. Typically an antisense oligo hybridizes with its target RNA in a cell and thereby leads to the inactivation of the target transcript. DNA and RNA oligos taken up by cells in culture are rapidly degraded unless they are chemically modified. Phosphorothioate substituted DNA oligos are resistant to nucleases and upon uptake into the cell inhibit gene expression by stimulating an RNase H activity which degrades the RNA component of the mRNA/DNA hybrid formed.
Oligo mixtures have been shown to be more effective in antisense studies than individual oligos (Nieto et al (1994) Science 264:835–839; Morgan et al (1993) Nucleic Acids Res. 21:4615–4620; Dattagupta et al (1998) U.S. Pat. No. 5,739,309) but are not widely used as they are not as readily synthesized. Mixtures of oligos are effective at much lower concentrations and with reduced toxicity. The observations suggest that the rate limiting step for antisense inhibition is the interaction of the oligo with its target mRNA. Mixtures of antisense oligos by sensing a larger mRNA target may be more efficient and thus require lower inhibitory concentrations. An additional advantage of oligo mixtures is reduced toxicity through reduced concentrations and increased specificity. It has been suggested that non-specific effects may result from short stretches of homology between an oligo and another non-target mRNA molecule that may hybridize long enough to be destroyed by RNase H (Wolf (1992) PNAS 89:7305–7309). The use of mixtures of oligos corresponding to different parts of the mRNA to be inactivated should reduce these effects since no one oligo would accumulate to a significant extent leaving the target mRNA as the site of preferred hybridization.
The present invention provides a means for the facile production of phosphorothioate containing oligo mixtures, and DNA or RNA polymers. The method involves the use of bacteria as an economical means to generate milligram quantities of DNase resistant oligo mixtures. Large fermentors could presumably be used to synthesize gram quantities of dsDNA, ssDNA, or oligo mixtures. The nucleic acids generated are stereospecific, corresponding to the preferred more stable Rp configuration for antisense work. To produce antisense oligo mixtures no DNA sequence information is required, simply the orientation of the cloned insert with respect to transcription. For example, cDNA clones that have been inserted unidirectionally into a recombinant vector (Alting-Meese and Short (1989) Nucleic Acids Res. 17:9494–9501) can be manipulated to generate antisense oligos.